Infection prophylaxis using immune response modifier compounds

ABSTRACT

The present invention provides methods of providing prophylaxis to a subject against an infectious agent. In general, the methods include topically administering to the respiratory tract of a subject an IRM compound in an amount effective to reduce infection by the agent.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application Ser.No. 60/493,109, filed Aug. 5, 2003.

BACKGROUND

Immune response modifiers (“IRMs”) include compounds that possess potentimmunomodulating activity including but not limited to antiviral andantitumor activity. Certain IRMs affect immune activity by modulatingthe production and secretion of cytokines. For example, cytokines thatare induced by certain small molecule IRM compounds include but are notlimited to Type I interferons, TNF-α, IL-1, IL-6, IL-8, IL-10, IL-12,MIP-1, and MCP-1. Alternatively, certain IRM compounds can inhibitproduction and secretion of certain T_(H)2 cytokines such as IL-4 andIL-5.

By stimulating certain aspects of the immune system, suppressing otheraspects, or both, IRMs may be used to treat many diseases andconditions. For example, IRM compounds may be useful for treating viraldiseases, neoplasias, fungal diseases, neoplastic diseases, parasiticdiseases, atopic diseases, and opportunistic infections and tumors thatoccur after suppression of cell-mediated immunity. IRM compounds alsomay be useful for promoting healing of wounds and post-surgical scars.Specifically, but not exclusively, diseases that may be treated usingIRM compounds include, but are not limited to, external genital andperianal warts caused by human papillomavirus, basal cell carcinoma,eczema, essential thrombocythaemia, hepatitis B, multiple sclerosis,neoplastic diseases, atopic dermatitis, asthma, allergies, psoriasis,rheumatoid arthritis, type I herpes simplex, and type II herpes simplex.

Certain formulations of a small molecule imidazoquinoline IRM compoundhave been shown to be useful for the therapeutic treatment of certaincancerous or pre-cancerous lesions (See, e.g., Geisse et al., J. Am.Acad. Dermatol., 47(3): 390-398 (2002); Shumack et al., Arch. Dermatol.,138: 1163-1171 (2002); U.S. Pat. No. 5,238,944; and WO 03/045391).

IRM compounds also can modulate humoral immunity by stimulating antibodyproduction by B cells. Further, various IRMs have been shown to beuseful as vaccine adjuvants (see, e.g., U.S. Pat. Nos. 6,083,505 and6,406,705).

Certain IRM compounds are known to be agonists of at least one Toll-likereceptor (TLR). For example, IRM compounds are known to be an agonist ofTLR6, TLR7, TLR8, or some combination thereof. Also, for example,certain modified oligonucleotide IRM compounds are known to be agonistsof TLR9.

SUMMARY

It has been found that certain IRMs can be used to provide prophylaxisagainst an infectious agent when topically administered to therespiratory tract of a subject. Moreover, infection prophylaxis may beprovided regardless of whether the IRM is administered before or afterthe subject is exposed to the infectious agent.

Accordingly, the present invention provides a method of providingprophylaxis to a subject against an infectious agent. The methodincludes topically administering to the respiratory tract of a subjectan IRM compound in an amount effective to limit infection by the agent.In some embodiments, the IRM compound can be administered from about 72hours prior to exposure to the infectious agent to about 72 hours afterexposure to the infectious agent.

Various other features and advantages of the present invention shouldbecome readily apparent with reference to the following detaileddescription, examples, claims and appended drawings. In several placesthroughout the specification, guidance is provided through lists ofexamples. In each instance, the recited list serves only as arepresentative group and should not be interpreted as an exclusive list.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph comparing viral titers in rats after treatmentwith vehicle, IFN-α, or IRM compound four hours before viral challenge.

FIG. 2 is a bar graph comparing viral titers in rats after treatmentwith vehicle, IFN-α, or IRM compound twenty-four hours and again at fourhours before viral challenge.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS OF THE INVENTION

The present invention relates to methods of providing prophylaxisagainst an infectious agent by topically administering an IRM compoundto the respiratory system of a subject. In some embodiments of thepresent invention, the IRM compound is administered in a relativelylimited dosing regimen that is specifically initiated upon actual,suspected, or anticipated possible exposure to an infectious agent.Therefore, certain methods of the present invention may be particularlysuited for providing infection prophylaxis for those who have been,intend to be, or suspect that they may be exposed to an infectiousagent.

For purposes of this invention, the following terms shall have themeanings set forth as follows:

“Agonist” refers to a compound that can combine with a receptor (e.g., aTLR) to induce a cellular activity. An agonist may be a ligand thatdirectly binds to the receptor. Alternatively, an agonist may combinewith a receptor indirectly by, for example, (a) forming a complex withanother molecule that directly binds to the receptor, or (b) otherwiseresults in the modification of another compound so that the othercompound directly binds to the receptor. An agonist may be referred toas an agonist of a particular TLR (e.g., a TLR6 agonist) or a particularcombination of TLRs (e.g., a TLR 7/8 agonist—an agonist of both TLR7 andTLR8).

“Cellular activity” refers to a biological activity (e.g., cytokineproduction), the modulation of which results from an agonist-receptorinteraction or an antagonist-receptor interaction.

“Exposure” of a subject to a pathogen refers to actual or anticipatedcontact between the subject and the pathogen. “Actual exposure” refersto exposure in fact, whether known or unknown. “Anticipated exposure”refers to any level of expected possibility of being exposed to apathogen.

“Induce” and variations thereof refer to any measurable increase incellular activity. Thus, as used herein, “induce” or “induction” may bereferred to as a percentage of a normal level of activity. In the caseof a cell product such as, for example, a cytokine, chemokine, orco-stimulatory marker, “induce” also may refer to any measurableincrease in the amount of the cell product that is produced in responseto a stimulus.

“Prophylaxis” refers to any degree of limiting an infection by aninfectious agent including (a) preventing or limiting an initialinfection, (b) preventing or limiting the spread of an existinginfection, or both. “Prophylaxis” may be used interchangeably with“reduce infection” and variations thereof.

“Respiratory Tract” refers, generally, to the major structures andpassages that permit or provide air flow between the environment and thelungs of a subject. “Upper Respiratory Tract” refers, generally, to thenasal cavity, paranasal sinuses, nasopharynx, oral cavity, pharynx, andlarynx. “Lower Respiratory Tract” refers, generally, to the trachea andlungs, including the bronchi, bronchioles, and alveoli.

“TLR-mediated” refers to a biological or biochemical activity thatresults, directly or indirectly, from TLR function. A particularbiological or biochemical activity may be referred to as mediated by aparticular TLR (e.g., “TLR6-mediated” or “TLR7-mediated”).

“Topical” refers to administering the IRM compound to a surface of therespiratory tract. Topical administration to the respiratory tract canoccur via formulations including but not limited to an aerosol, anon-aerosol spray, a cream, an ointment, a gel, a lotion, a mouthwash,and the like.

The methods of the present invention can provide general prophylaxisagainst infection by an infectious agent. One feature of certain methodsof the present invention is that topical administration of the IRMcompound can provide infection prophylaxis against one or a plurality ofknown or unknown infectious agents. Knowledge or even suspicion of theidentity of the particular infectious agent to which one has or might beexposed is not required. Accordingly, the methods of the presentinvention may be particularly useful when exposure to an infectiousagent has occurred, is suspected to have occurred, or is anticipated,but the identity of the particular infectious agent is not known. Eventsin which the methods of the present invention thus may be particularlyuseful include, but are not limited to, outbreaks of new or previouslyunidentified infectious agents, biological warfare, bio-terrorism, andexposure to environments that can have a relatively large number ofdifferent infectious agents (e.g., health care clinics, daycare centers,and the like).

In certain embodiments, it may be possible to enhance infectionprophylaxis against a particular infectious agent, if known, bycombining administration of the IRM compound with separate orco-administration of one or more components of the infectious agent.However, infection prophylaxis using the methods of the presentinvention does not require such combinations or—as stated above—evenknowledge of the identity of the infectious agent. Another feature ofcertain embodiments of the present invention is the ease and relativelylimited discomfort associated with administration of the IRM compound.Topical administration to the respiratory tract can involve inhalationof an aerosol or non-aerosol formulation, the inhaled formulation beingdeposited on the surface of structures of the upper respiratory tract,the lower respiratory tract, or both.

Alternatively, topical administration of the IRM compound to therespiratory tract may involve contacting a surface of the respiratorytract—typically, the upper respiratory tract—with a cream, gel,mouthwash, or the like. The formulation may be left in place followingadministration (e.g., a cream or gel applied to a surface of the oral ornasal cavity) or may be discarded (e.g., a mouthwash). Administration ofthe IRM compound can be minimally invasive, particularly when comparedto subcutaneous, intramuscular, or transdermal vaccination.

Yet another feature of certain methods of the present invention is thatadministration of the IRM compound can be temporally connected to anexposure event, whether the exposure event has occurred, is suspected ofhaving occurred, or is expected to occur. Moreover, infectionprophylaxis can be provided without requiring administration of aprophylactic agent over weeks or months of treatment. For example, insome embodiments, infection prophylaxis can be provided by one or twodoses of IRM compound administered about 72 hours or less before ananticipated exposure to an infectious agent. In some embodiments,infection prophylaxis can be provided by a single dose of IRM compoundadministered four hours before exposure to the infectious agent.

Alternatively, infection prophylaxis can be provided by administeringthe IRM compound after exposure to the infectious agent has occurred oris suspected to have occurred. For example, in some embodiments,infection prophylaxis can be provided by one or two doses of IRMcompound administered about 72 hours or less after an actual orsuspected exposure event. In certain embodiments, infection prophylaxiscan be provided with a single dose of IRM compound provided within 24hours of the exposure event. In many embodiments, infection prophylaxiscan be provided without requiring administration of a prophylactic ortherapeutic agent over weeks or months of treatment.

In certain embodiments, administration of IRM compound before ananticipated exposure event may be combined with administration of IRMcompound after an actual or suspected exposure event.

The features of certain embodiments of the present invention can beparticularly desirable for some applications. For example, each of: (a)the temporal connection to an exposure event, (b) ease ofadministration, and (c) relatively short dosing regimens—alone and invarious combinations—can increase the likelihood and extent ofcompliance, thereby increasing the efficacy of the prophylaxis.

As another example, the general infection prophylaxis conferred byadministering IRM compound renders certain embodiments useful forproviding infection prophylaxis even when the number and identity ofinfectious agent(s) is unknown. Thus, methods of the present inventionmay be particularly desirable in circumstances when, at the time when astandard vaccination would have to be given to provide infectionprophylaxis, it is unclear to which, if any, infectious agents one maybe exposed. Thus, infection prophylaxis using methods of the presentinvention can be provided on an “as needed” basis, thereby reducing thenumber of vaccinations administered unnecessarily.

Additionally, a single course of administering IRM compound may provideat least as great a scope—and in some cases, even greater scope—ofinfection prophylaxis as several vaccinations, with reduced costs (e.g.,less developmental cost, one administration versus several, etc.), lessdiscomfort, and without the inherent risk associated with standardvaccinations. All of the above factors may contribute to increasedcompliance compared to traditional vaccinations, which, in turn, mayresult in a higher percentage of individuals in a treated populationhaving efficacious infection prophylaxis.

Infectious agents against which IRM compounds may provide infectionprophylaxis using methods of the present invention include, but are notlimited to: (a) viruses such as, for example, variola, HIV, CMV, VZV,rhinovirus, adenovirus, coronavirus (including, e.g., SARS), influenza,para-influenza, ebola, hepatitis B, hepatitis C, and West Nile virus;(b) bacteria, such as those that can cause tuberculosis, anthrax,listeriosis, and leprosy; (c) other infectious agents such as prions andparasites (e.g., Leishmania spp.);

For example, certain methods of the present invention may beparticularly useful for daycare or health care workers who have been orwill be exposed to an infectious agent. Thus, certain methods of thepresent invention may secondarily reduce sick time taken by employees incertain industries.

As another example, certain methods of the present invention may beparticularly useful for providing prophylaxis against biological warfareor bio-terrorism agents. Such methods may be employed by militarypersonnel either (a) before an anticipated exposure to a biologicalwarfare agent, (b) before entering an area where a biological warfareagent has previously been used, or (c) after a suspected or knownexposure to a biological warfare agent. Certain methods of the presentinvention may be particularly useful in the event of exposure to abio-terrorism agent. In some cases, the methods may be employed byemergency personnel including police, medical personnel, firefighters,and the like. In other cases, the methods may be employed by the generalpopulation or a subset of the general population. In either case,certain methods may provide infection prophylaxis when employed duringor after either (a) exposure to a bio-terrorism agent, or (b) a warningthat a bio-terrorism event may occur.

As yet another example, the invention may be practiced to provideinfection prophylaxis for those who must—or choose to—travel to an areaexperiencing an outbreak of an infectious agent. For example, healthcare workers may travel to an area experiencing such an outbreak to carefor those affected by the infectious agent. Others may choose not tocancel or delay business or pleasure travel plans. In these cases,practicing the invention may help provide infection prophylaxis to thosetraveling into the area of an outbreak, thereby, for example,facilitating health care for those affected, minimizing the economic andsocial cost of the outbreak, or both.

Type 1 interferons such as, for example, IFN-α have been shown topossess antiviral activity, perhaps by induction of a nonspecificantiviral state. Administration of IFN-α can be effective, as amonotherapy, preventing some viral infections. However, IFN-αmonotherapy has not gained widespread acceptance, perhaps at least inpart due to associated side-effects such as, for example, nasalstuffiness, increased sneezing, bleeding, and nasal erosion.

FIGS. 1 and 2 summarize data demonstrating the efficacy of the inventionin providing infection prophylaxis against viral infection.Administration of the IRM compound prior to intranasal infection withinfluenza virus dramatically reduced the viral titer present in the lungand nasal cavity 24 hours after the infection, even compared topretreatment with IFN-α.

Table 3 summarizes data that demonstrates the efficacy of IRM compoundpreventing replication of various viruses. IRM compound was used toinhibit viral replication in each of two ways: directly and indirectly.Indirect inhibition of viral replication occurred when IRM compound wasused to stimulate a culture of peripheral blood mononuclear cells(PBMCs). Factors secreted by the PBMCs into the culture supernatant werecollected and tested for the ability to inhibit viral replication.

Inhibition of viral replication occurred with PBMC supernatant solutionsat approximate EC₅₀ values of 0.17 μM against coronavirus, <0.0015 μMagainst influenza A/Panama/2007/99, <0.0015 μM against influenza A/NewCalcdonia/20/99, and 0.15 μM against SARS.

Direct inhibition of viral replication occurred when IRM compound itselfwas tested for the ability to inhibit viral replication. IRM compoundalone added to media was able to inhibit SARS replication with anapproximate EC₅₀ at 3 μM.

The results of these studies indicate that IRM compounds stimulatesignificant antiviral factors to be secreted from PBMCs into supernatantsolutions. When considered together with the data from FIGS. 1 and 2,the antiviral properties associated with practicing the invention aregreater than the antiviral properties of IFN-α monotherapy. These datasupport the use of IRM compounds for providing non-specific prophylacticand/or therapeutic treatment of viral infections.

Immune response modifiers (“IRMs”) include compounds that possess potentimmunomodulating activity including but not limited to antiviral andantitumor activity. Certain IRMs modulate the production and secretionof cytokines. For example, certain IRM compounds induce the productionand secretion of cytokines such as, e.g., Type I interferons, TNF-α,IL-1, IL-6, IL-8, IL-10, IL-12, MIP-1, and/or MCP-1. As another example,certain IRM compounds can inhibit production and secretion of certainT_(H)2 cytokines, such as IL-4 and IL-5. Additionally, some IRMcompounds are said to suppress IL-1 and TNF (U.S. Pat. No. 6,518,265).

Certain IRMs are small organic molecules (e.g., molecular weight underabout 1000 Daltons, preferably under about 500 Daltons, as opposed tolarge biological molecules such as proteins, peptides, and the like)such as those disclosed in, for example, U.S. Pat. Nos. 4,689,338;4,929,624; 4,988,815; 5,037,986; 5,175,296; 5,238,944; 5,266,575;5,268,376; 5,346,905; 5,352,784; 5,367,076; 5,389,640; 5,395,937;5,446,153; 5,482,936; 5,693,811; 5,741,908; 5,756,747; 5,939,090;6,039,969; 6,083,505; 6,110,929; 6,194,425; 6,245,776; 6,331,539;6,376,669; 6,451,810; 6,525,064; 6,541,485; 6,545,016; 6,545,017;6,558,951; 6,573,273; 6,656,938; 6,660,735; 6,660,747; 6,664,260;6,664,264; 6,664,265; 6,667,312; 6,670,372; 6,677,347; 6,677,348;6,677,349; 6,683,088; 6,756,382; European Patent 0 394 026; U.S. PatentPublication Nos. 2002/0016332; 2002/0055517; 2002/0110840; 2003/0133913;2003/0199538; and 2004/0014779; and International Patent PublicationNos. WO 01/74343; WO 02/46749 WO 02/102377; WO 03/020889; WO 03/043572;WO 03/045391; WO 03/103584; and WO 04/058759.

Additional examples of small molecule IRMs include certain purinederivatives (such as those described in U.S. Pat. Nos. 6,376,501, and6,028,076), certain imidazoquinoline amide derivatives (such as thosedescribed in U.S. Pat. No. 6,069,149), certain imidazopyridinederivatives (such as those described in U.S. Pat. No. 6,518,265),certain benzimidazole derivatives (such as those described in U.S. Pat.No. 6,387,938), certain derivatives of a 4-aminopyrimidine fused to afive membered nitrogen containing heterocyclic ring (such as adeninederivatives described in U.S. Pat. Nos. 6,376,501; 6,028,076 and6,329,381; and in WO 02/08905), and certain3-p-D-ribofuranosylthiazolo[4,5-d]pyrimidine derivatives (such as thosedescribed in U.S. Publication No. 2003/0199461).

Other IRMs include large biological molecules such as oligonucleotidesequences. Some IRM oligonucleotide sequences contain cytosine-guaninedinucleotides (CpG) and are described, for example, in U.S. Pat. Nos.6,194,388; 6,207,646; 6,239,116; 6,339,068; and 6,406,705. SomeCpG-containing oligonucleotides can include synthetic immunomodulatorystructural motifs such as those described, for example, in U.S. Pat.Nos. 6,426,334 and 6,476,000. Other IRM nucleotide sequences lack CpGsequences and are described, for example, in International PatentPublication No. WO 00/75304.

Other IRMs include biological molecules such as aminoalkyl glucosaminidephosphates (AGPs) and are described, for example, in U.S. Pat. Nos.6,113,918; 6,303,347; 6,525,028; and 6,649,172.

The IRM compound may be any suitable IRM compound. In some embodimentsof the present invention, the IRM compound may include a 2-aminopyridinefused to a five membered nitrogen-containing heterocyclic ring, or a4-aminopyrimidine fused to a five membered nitrogen-containingheterocyclic ring. In some embodiments, suitable IRM compounds includebut are not limited to the small molecule IRM compounds (e.g., molecularweight of less than about 1000 Daltons) described above.

Certain small molecule IRM compounds—those having a 2-aminopyridinefused to a five membered nitrogen-containing heterocyclic ring—include,for example, imidazoquinoline amines including but not limited tosubstituted imidazoquinoline amines; tetrahydroimidazoquinoline aminesincluding but not limited to amide substitutedtetrahydroimidazoquinoline amines, sulfonamide substitutedtetrahydroimidazoquinoline amines, urea substitutedtetrahydroimidazoquinoline amines, aryl ether substitutedtetrahydroimidazoquinoline amines, heterocyclic ether substitutedtetrahydroimidazoquinoline amines, amido ether substitutedtetrahydroimidazoquinoline amines, sulfonamido ether substitutedtetrahydroimidazoquinoline amines, urea substitutedtetrahydroimidazoquinoline ethers, and thioether substitutedtetrahydroimidazoquinoline amines; imidazopyridine amines including butnot limited to amide substituted imidazopyridine amines, sulfonamidesubstituted imidazopyridine amines, urea substituted imidazopyridineamines, aryl ether substituted imidazopyridine amines, heterocyclicether substituted imidazopyridine amines, amido ether substitutedimidazopyridine amines, sulfonamido ether substituted imidazopyridineamines, urea substituted imidazopyridine ethers, and thioethersubstituted imidazopyridine amines; 1,2-bridged imidazoquinoline amines;6,7-fused cycloalkylimidazopyridine amines; imidazonaphthyridine amines;tetrahydroimidazonaphthyridine amines; oxazoloquinoline amines;thiazoloquinoline amines; oxazolopyridine amines; thiazolopyridineamines; oxazolonaphthyridine amines; thiazolonaphthyridine amines; and1H-imidazo dimers fused to pyridine amines, quinoline amines,tetrahydroquinoline amines, naphthyridine amines, ortetrahydronaphthyridine amines.

In certain embodiments, the IRM compound may be an imidazonaphthyridineamine, a tetrahydroimidazonaphthyridine amine, an oxazoloquinolineamine, a thiazoloquinoline amine, an oxazolopyridine amine, athiazolopyridine amine, an oxazolonaphthyridine amine, or athiazolonaphthyridine amine.

In certain embodiments, the IRM compound may be a substitutedimidazoquinoline amine, a tetrahydroimidazoquinoline amine, animidazopyridine amine, a 1,2-bridged imidazoquinoline amine, a 6,7-fusedcycloalkylimidazopyridine amine, an imidazonaphthyridine amine, atetrahydroimidazonaphthyridine amine, an oxazoloquinoline amine, athiazoloquinoline amine, an oxazolopyridine amine, a thiazolopyridineamine, an oxazolonaphthyridine amine, or a thiazolonaphthyridine amine.

As used herein, a substituted imidazoquinoline amine refers to an amidesubstituted imidazoquinoline amine, a sulfonamide substitutedimidazoquinoline amine, a urea substituted imidazoquinoline amine, anaryl ether substituted imidazoquinoline amine, a heterocyclic ethersubstituted imidazoquinoline amine, an amido ether substitutedimidazoquinoline amine, a sulfonamido ether substituted imidazoquinolineamine, a urea substituted imidazoquinoline ether, a thioethersubstituted imidazoquinoline amines, or a 6-, 7-, 8-, or 9-aryl orheteroaryl substituted imidazoquinoline amine. As used herein,substituted imidazoquinoline amines specifically and expressly exclude1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine and4-amino-α,α-dimethyl-2-ethoxymethyl-1H-imidazo[4,5-c]quinolin-1-ethanol.

In certain embodiments, the IRM compound can be a sulfonamidesubstituted imidazoquinoline amine. In certain specific embodiments, theIRM compound can beN-{2-[4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl]-1,1-dimethylethyl}methanesulfonamide.

Suitable IRM compounds also may include the purine derivatives,imidazoquinoline amide derivatives, benzimidazole derivatives, adeninederivatives, and oligonucleotide sequences described above.

In some embodiments, the IRM compound may be a compound identified as anagonist of one or more TLRs. In some embodiments of the presentinvention, the IRM compound may be an agonist of at least one TLR,preferably an agonist of TLR6, TLR7, or TLR8. The IRM compound may, insome cases, be an agonist of TLR 9. In certain embodiments, the IRMcompound may be an agonist of at least TLR7—i.e., may be, for example, aTLR7/8 agonist or a TLR7-selective agonist.

As used herein, the term “TLR7/8 agonist” refers to a compound that actsas an agonist of both TLR7 and TLR8. A “TLR7-selective agonist” refersto a compound that acts as an agonist of TLR7, but does not act as anagonist of TLR8. Thus, a “TLR7-selective agonist” may refer to acompound that acts as an agonist for TLR7 and for no other TLR, but itmay alternatively refer to a compound that acts as an agonist of TLR7and a TLR other than TLR8 such as, for example, TLR6.

The TLR agonism for a particular compound may be assessed in anysuitable manner. For example, assays for detecting TLR agonism of testcompounds are described, for example, in U.S. patent application Ser.No. 10/732,563, filed Dec. 10, 2003, and recombinant cell lines suitablefor use in such assays are described, for example, in U.S. patentapplication Ser. No. 10/732,796, filed Dec. 10, 2003.

Regardless of the particular assay employed, a compound can beidentified as an agonist of a particular TLR if performing the assaywith a compound results in at least a threshold increase of somebiological activity mediated by the particular TLR. Conversely, acompound may be identified as not acting as an agonist of a specifiedTLR if, when used to perform an assay designed to detect biologicalactivity mediated by the specified TLR, the compound fails to elicit athreshold increase in the biological activity. Unless otherwiseindicated, an increase in biological activity refers to an increase inthe same biological activity over that observed in an appropriatecontrol. An assay may or may not be performed in conjunction with theappropriate control. With experience, one skilled in the art may developsufficient familiarity with a particular assay (e.g., the range ofvalues observed in an appropriate control under specific assayconditions) that performing a control may not always be necessary todetermine the TLR agonism of a compound in a particular assay.

The precise threshold increase of TLR-mediated biological activity fordetermining whether a particular compound is or is not an agonist of aparticular TLR in a given assay may vary according to factors known inthe art including but not limited to the biological activity observed asthe endpoint of the assay, the method used to measure or detect theendpoint of the assay, the signal-to-noise ratio of the assay, theprecision of the assay, and whether the same assay is being used todetermine the agonism of a compound for both TLRs. Accordingly it is notpractical to set forth generally the threshold increase of TLR-mediatedbiological activity required to identify a compound as being an agonistor a non-agonist of a particular TLR for all possible assays. Those ofordinary skill in the art, however, can readily determine theappropriate threshold with due consideration of such factors.

Assays employing HEK293 cells transfected with an expressible TLRstructural gene may use a threshold of, for example, at least athree-fold increase in a TLR-mediated biological activity (e.g., NFKBactivation) when the compound is provided at a concentration of, forexample, from about 1 μM to about 10 μM for identifying a compound as anagonist of the TLR transfected into the cell. However, differentthresholds and/or different concentration ranges may be suitable incertain circumstances. Also, different thresholds may be appropriate fordifferent assays.

The IRM compound may be provided in a formulation suitable for topicaladministration to the respiratory tract of a subject. The IRM compoundmay be provided in any suitable form including but not limited to asolution, a suspension, an emulsion, a dry powder, or any form ofmixture. The IRM compound may be administered in formulation with anypharmaceutically acceptable excipient, carrier, or vehicle. Theformulation may be administered in any conventional dosage form fortopical delivery to a surface of the respiratory tract. Such dosageforms include but are not limited to an aerosol formulation, anon-aerosol spray, a cream, an ointment, a gel, a lotion, a mouthwash,and the like. Suitable aerosol formulations are described, for example,in U.S. Pat. No. 6,126,919. Alternative formulations are described, forexample, in U.S. Pat. No. 5,238,944; EP 0 394 026; U.S. Pat. No.6,365,166; and U.S. Pat. No. 6,245,776. The formulation may furtherinclude one or more additives including but not limited to adjuvants,penetration enhancers, colorants, fragrances, moisturizers, thickeners,and the like.

In some embodiments, the methods of the present invention includeadministering IRM compound to a subject in a formulation of, forexample, from about 0.001% to about typically 10% (unless otherwiseindicated, all percentages provided herein are weight/weight withrespect to the total formulation) to the subject, although in someembodiments the IRM compound may be administered using a formulationthat provides IRM compound in a concentration outside of this range. Incertain embodiments, the method includes administering to a subject aformulation that includes from about 0.01% to about 1.0% IRM compound,for example, a formulation that includes about 0.375% IRM compound.

An amount of an IRM compound effective for providing infectionprophylaxis is an amount sufficient to either prevent or limit (a) aninitial infection, or (b) the spread of an existing infection, or both.One method of determining an amount effective for providing infectionprophylaxis is to determine an amount effective to reduce—to astatistically significant extent—nasal or lung titers of the infectiousagent 24 hours after the latter of: (a) exposure to the infectiousagent, or (b) administration of the IRM compound.

The precise amount of IRM compound in a dose may vary according tofactors known in the art including but not limited to the physical andchemical nature of the IRM compound, the nature of the carrier, theintended dosing regimen, the state of the subject's immune system (e.g.,suppressed, compromised, stimulated), the method of administering theIRM compound, the species to which the formulation is beingadministered, and the infectious agent or agents, if known, to which thesubject has been or is expected to be exposed. Accordingly it is notpractical to set forth generally the amount that constitutes an amountof IRM compound effective for providing infection prophylaxis for allpossible applications. Those of ordinary skill in the art, however, canreadily determine the appropriate amount with due consideration of suchfactors.

In some embodiments, the methods of the present invention includeadministering sufficient IRM compound to provide a dose of, for example,from about 100 ng/kg to about 50 mg/kg to the subject, although in someembodiments the methods may be performed by administering IRM compoundin concentrations outside this range. In some of these embodiments, themethod includes administering sufficient IRM compound to provide a doseof from about 10 μg/kg to about 5 mg/kg to the subject, for example, adose of from about 100 μg/kg to about 1 mg/kg.

The dosing regimen may depend at least in part on many factors known inthe art including but not limited to the physical and chemical nature ofthe IRM compound, the nature of the carrier, the amount of IRM compoundbeing administered, the state of the subject's immune system (e.g.,suppressed, compromised, stimulated), the method of administering theIRM compound, the species to which the formulation is beingadministered, and the infectious agent or agents, if known, to which thesubject has been or is expected to be exposed. Accordingly it is notpractical to set forth generally the dosing regimen effective to provideinfection prophylaxis for all possible applications. Those of ordinaryskill in the art, however, can readily determine the appropriate amountwith due consideration of such factors.

In some embodiments of the invention, the IRM compound may beadministered, for example, from once to about twelve times over theentire course of treatment, although in some embodiments the methods ofthe present invention may be performed by administering the IRM compoundat a frequency outside this range. In certain embodiments, the IRMcompound may be administered from once to about four times over theentire course of treatment. In one particular embodiment, the IRMcompound is administered once. In another particular embodiment, the IRMcompound is administered twice.

The IRM compound may be administered before an anticipated exposure tothe infectious agent. In some embodiments, the IRM compound isadministered once per day for a period before an anticipated exposure tothe infectious agent. In certain embodiments, the IRM compound may beadministered once per day for two days. In other embodiments, the IRMcompound may be administered in a single dose. In some embodiments, atleast one dose of the IRM compound is administered 72 hours or lessbefore an anticipated exposure to the infectious agent. In oneparticular embodiment, for example, the IRM compound is administered ina single dose, about 4 hours prior to exposure to the infectious agent.

In alternative embodiments, the IRM compound may be administered after asuspected or confirmed exposure to the infectious agent. In theseembodiments, the IRM compound may be administered once per day for aperiod after a suspected or actual exposure to the infectious agent. Incertain embodiments, the IRM compound may be administered once per dayfor two days. In other embodiments, the IRM compound may be administeredin a single dose. In some embodiments, at least one dose of the IRMcompound is administered 72 hours or less after the actual or suspectedexposure to the infectious agent. In one particular embodiment, forexample, the IRM compound is administered in a single dose, about 4hours after exposure to the infectious agent.

In embodiments in which the IRM compound may be administered afteractual or suspected exposure to the infectious agent, administration ofthe IRM can begin prior to the detectable onset of symptoms resultingfrom infection by the infectious agent. In such embodiments, the courseof treatment may conclude before the onset of any symptoms.Alternatively, in some embodiments, the course of treatment may beginprior to the onset of symptoms and continue even after the subjectexperiences symptoms resulting from infection by the infectious agent.In such cases, it is expected that the extent of infection will besignificantly reduced by practicing the methods of the invention,thereby limiting the severity, extent, and/or duration of symptoms.

In other alternative embodiments, the IRM compound may be administeredbefore an anticipated exposure to an infectious agent, and again afterthe exposure to the infectious agent has, or is suspected to have,occurred. Again, in such embodiments, the course of treatment mayconclude prior to, or may continue after, the onset of any symptomsresulting from infection by the infectious agent.

The methods of the present invention may be performed on any suitablesubject. Suitable subjects include but are not limited to animals suchas but not limited to humans, non-human primates, rodents, dogs, cats,horses, pigs, sheep, goats, or cows.

EXAMPLES

The following examples have been selected merely to further illustratefeatures, advantages, and other details of the invention. It is to beexpressly understood, however, that while the examples serve thispurpose, the particular materials and amounts used as well as otherconditions and details are not to be construed in a matter that wouldunduly limit the scope of this invention. Unless otherwise provided, allpercentages are given as w/w %.

The IRM compound used in the examples isN-{2-[4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl]-1,1-dimethylethyl}methanesulfonamide,the synthesis of which is described in example 268 of U.S. Pat. No.6,677,349.

Example 1

IRM compound was prepared as a 0.375% solution formulation capable ofbeing nasally administered via a spray pump. The formulation vehicle wasprepared as follows: TABLE 1 Excipient w/w % Carboxymethyl cellulosesodium, low viscosity, USP 0.1 (Spectrum Chemicals and LaboratoryProducts, Inc., Gardena, CA,) Benzalkonium chloride, Ph. Eur. (Fluka,Buchs Switzerland) 0.02 Disodium EDTA, USP (Spectrum Chemicals) 0.1L-Lactic acid, Purac (Lincolnshire, IL) 1.53 PEG 400, NF (SpectrumChemicals) 15 1 N NaOH, NF (Spectrum Chemicals) qs Water qs Total 100.00pH 4.0

Carboxymethyl cellulose sodium, low viscosity, (CMC) was hydrated inwater (about 50% of total) for 20 minutes with stirring. The EDTA wasadded and dissolved. The CMC/EDTA solution was mixed with thebenzalkonium chloride to form a CMC/EDTA/BAC solution. Separately, thelactic acid and PEG 400 were mixed with water. For the IRM formulation,IRM compound was dissolved into the lactic acid/PEG 400 solution. TheCMC/EDTA/BAC solution was mixed with lactic acid/PEG 400 solution toprepare the Vehicle formulation. The CMC/EDTA/BAC solution was mixedwith lactic acid/PEG 400/IRM solution to prepare the IRM formulation. 1N NaOH was added, as necessary, to adjust each formulation to a pH of4.0. Finally, water was added to each formulation to adjust to the finalformulation weight.

Example 2

Fisher 344 rats (Charles River Laboratories, Raleigh, N.C.) were dividedinto six treatment groups. Rats in each group were infected intranasallywith humanized, non-lethal influenza virus. 24 hours after infection,viral titers were measured in nasal lavage fluid and whole lunghomogenates. The influenza virus and methods for measuring viral titersare described in Burleson, Gary L., “Influenza Virus Host ResistanceModel for Assessment of Immunotoxicity, Immunostimulation, and AntiviralCompounds,” Methods in Immunology 2: 181-202, Wiley-Liss Inc., 1995.

Each of the six treatment groups received a different pre-infectiontreatment. Rats in each group received the treatment indicated in Table2. The results are summarized in FIG. 1 and FIG. 2. TABLE 2 GroupTreatment 1 Vehicle formulation (Table 1), 50 μL (25 μL per nare), 1x* 2Interferon-α (rat recombinant IFN-α, Cat. No. PRP13, Serotec Inc.,Raleigh, NC), 10,000 IU, 1x 3 IRM formulation (Table 1), 50 μL (25 μLper nare), 1x 4 Vehicle formulation (Table 1), 50 μL (25 μL per nare),2x** 5 Interferon-α, 10,000 IU, 2x (Day −1: Product No. RR2030U, PierceBiotechnology, Inc., Rockford, IL; Day 0: Serotec Inc. Cat. No. PRP13) 6IRM formulation (Table 1), 50 μL (25 μL per nare), 2x*1x: one dose of treatment provided four hours before viral infection.**2x: one dose of treatment 24 hours (Day −1) before viral infection,second treatment four hours before viral infection (Day 0).

Example 3

Thirty-five mL of blood from volunteer donors was layered over 15 mL ofroom temperature Histopaque-1077 Hybri-Max (Sigma Chemical Co., St.Lois, Mo.) in an Accuspin tube (Sigma Chemical Co.) and centrifuged atroom temperature, 800×g for 15 minutes (Beckman centrifuge). PBMC band(2^(nd) layer) was removed and washed twice with Hanks Balanced SaltSolution (HBSS). Final cell pellet was resuspended in 10% Fetal BovineSerum and 1×Penicillin/Streptomycin in RPMI-1640. Suspension density wasdetermined using trypan blue and a hemacytometer. Density was brought to1×10⁶ cells/milliliter in growth media, 10 mLs was placed in a T25tissue culture flask, and suspension was allowed to equilibrate at 37°C. in a humidified incubator with 5-7% CO₂ for 1 hour.

IRM compound was added in growth media to desired final concentration.Supernatants were harvested after 24 hours and frozen until tested. Fiveconcentrations of IRM compound in test media or as PBMC supernatantsolutions (sup) were prepared for testing.

Viruses:

Human coronavirus (HcoV), strain OC43: Obtained from the American TypeCulture Collection (ATCC), Manassas, Va. Pools of virus made up in BSC-1cells.

Influenza A/Panama/2007/99 (H₃N₂): Obtained from the Centers for DiseaseControl and Prevention (CDC), Atlanta, Ga. Virus stocks were prepared inMDCK cells.

Influenza A/New Calcdonia/20/99 (H1N1): Obtained from the CDC, Atlanta,Ga. Virus stocks were prepared in MDCK cells.

Severe acute respiratory syndrome (SARS) coronavirus, strain Urbani.Obtained from the CDC, Atlanta, Ga. A pool was prepared in African greenmonkey kidney (Vero 76) cells.

Cells and Media:

BSC-1 cells (used for human corona viruses). Growth medium is MEM with5% fetal bovine serum (FBS) and 0.1% NaHCO₃. Test medium is MEM with 2%FBS, 0.1% NaHCO₃ and 50 μg/mL gentamycin.

MDCK cells (Maden Darby canine kidney, used for influenza virus),obtained from the ATCC. Growth medium is MEM with 5% FBS and 0.1%NaHCO₃. Test medium is MEM without serum, 0.18% NaHCO₃, 20 μgtrypsin/mL, 2.0 μg EDTA/mL, and 50 μg gentamycin/mL.

Vero 76 cells (used for SARS coronavirus), obtained from the ATCC.Growth medium is MEM with 5% fetal bovine serum (FBS) and 0.1% NaHCO₃.Test medium is MEM with 2% FBS, 0.1% NaHCO₃ and 50 μg gentamycin/mL.

Antiviral Testing Procedure:

For all viruses, cells were seeded to 96-well flat-bottomed tissueculture plates (Coming Glass Works, Corning, N.Y.), 0.2 mL/well, at theproper cell concentration, and incubated overnight at 37° C. in order toestablish a cell monolayer. When the monolayer was established, thegrowth medium was decanted. Each dilution of the compound was added in avolume of 0.1 mL to a total of 5 wells/dilution; approximately 5 minlater 0.1 mL of virus in test medium was added to 3 wells per dilution,and sterile virus diluent added to the remaining wells. Six wells wereexposed to drug diluent and virus to serve as virus controls, and 6wells were exposed to drug diluent and sterile virus diluent to serve asnormal cell controls. The plates were sealed with plastic wrap andincubated in a humidified incubator with 5% CO₂, 95% air atmosphere at37° C. for the time required for viral cytopathic effect (CPE) to fullydevelop in the virus control wells.

Cells were then examined microscopically for CPE, this being scored from0 (normal cells) to 4 (maximal, 100%, CPE). The cells in the toxicitycontrol wells were observed microscopically for morphologic changesattributed to cytotoxicity. This cytotoxicity was also graded at T (100%toxicity, complete cell sloughing from plate), PVH (60% cytotoxicity),P(40% cytotoxicity), PS (20% cytotoxicity), and 0 (normal cells). The50% effective dose (EC₅₀) and 50% cytotoxic dose (CC₅₀) were calculatedby regression analysis of the virus CPE data and the toxicity controldata, respectively. The selective index (SI) for each compound tested iscalculated by the formula: SI═CC₅₀÷EC₅₀.

After the plates were read visually for cytopathology and toxicity, thecells were then stained with neutral red to verify the visualdeterminations. To do this, 0.1 mL of sterile neutral red (0.034% inphysiological saline solution) was added to each well. The plates werewrapped in aluminum foil to eliminate light exposure and placed at 37°C. for one to two hours. All medium was removed and the cells gentlywashed 2×(0.2 mL/well for each wash) with phosphate buffered saline. Theplates were inverted and allowed to drain on a paper towel. Neutral redwas extracted from the cells by adding 0.2 mL of an equal volume mixtureof absolute ethanol and Sorensen's citrate buffer, pH4, to each well andplacing the plates in the dark at room temperature for 30 minutes. Thecontents of each well were mixed gently and the O.D. values of each wellwere obtained by reading the plates at 540 nm with a Model EL309microplate reader (Bio-Tek Instruments, Inc., Winooski, Vt.). The EC₅₀and CC₅₀ are calculated by regression analysis. The SI for each compoundtested was again calculated by the formula: SI═CC₅₀÷EC₅₀.

A known active substance was run in the same manner as above for eachbatch of compounds tested. For influenza viruses, ribavirin was used.For human coronavirus (HCOV) and SARS, ALFERON (Hemispherx Biopharma,Inc., Philadelphia, Pa.) was used. In these studies, selected cell typeswere pretreated with supernatant solutions from IRM-stimulated PBMCcultures or with media containing solubilized drug and then exposed todifferent types of respiratory viruses. Viral replication was thendetermined by viral cytopathic effect (CPE) analysis and neutral red(NR) analysis for confirmation. Cytotoxic concentrations were alsodetermined and were described by the concentration that produced 50%cell death. Antiviral activity was only considered to exist when theconcentration for 50% cytotoxicity was at least 3 times greater than theeffective concentration for 50% inhibition in the CPE assay (i.e, SI>3).Results are summarized in Table 3. TABLE 3 Virus Treatment CC₅₀ (μM)EC₅₀ (μM) SI (CC₅₀/EC₅₀) Influenza PBMC supernatant >1.5 <0.0015 >1000 A(H3N2) IRM >300 >300 0 Ribavirin >450 24 >19 Influenza PBMCsupernatant >1.5 <0.0015 >1000 A (H1N1) IRM >300 >300 0 Ribavirin >45014 >32 SARS PBMC supernatant >1.5 0.15 >10 IRM >150 >3 50ALFERON >32,000 <32 >1000 HCoV PBMC supernatant >1.5 0.17 >9IRM >150 >150 0 ALFERON >32,000 32 >1000

PBMC supernatant solutions from cells stimulated with IRM compound hadsignificant activity against SARS, both of the influenza viruses tested,and human coronavirus. IRM compound, alone, possessed significant directactivity against SARS.

The complete disclosures of the patents, patent documents andpublications cited herein are incorporated by reference in theirentirety as if each were individually incorporated. In case of conflict,the present specification, including definitions, shall control.

Various modifications and alterations to this invention will becomeapparent to those skilled in the art without departing from the scopeand spirit of this invention. Illustrative embodiments and examples areprovided as examples only and are not intended to limit the scope of thepresent invention. The scope of the invention is limited only by theclaims set forth as follows.

1. A method of providing prophylaxis to a subject against an infectiousagent comprising topically administering to the respiratory tract of asubject an IRM compound in an amount effective to reduce infection bythe agent.
 2. The method of claim 1 wherein the IRM compound isadministered nasally.
 3. The method of claim 1 wherein the IRM compoundis administered orally.
 4. The method of claim 1 wherein the IRMcompound is administered from about 72 hours prior to exposure to theinfectious agent to about 72 hours after exposure to the infectiousagent.
 5. The method of claim 4 wherein the IRM compound is administeredprior to and after exposure to the infectious agent.
 6. The method ofclaim 4 wherein the IRM compound is administered prior to but not afterexposure to the infectious agent.
 7. The method of claim 4 wherein theIRM compound is administered after but not prior to exposure to theinfectious agent.
 8. The method of claim 4 wherein the IRM compound isadministered in from 1 to about 12 doses.
 9. The method of claim 8wherein the IRM compound is administered in from 1 to about 3 doses. 10.The method of claim 9 wherein the IRM compound is administered in 1dose.
 11. The method of claim 10 wherein the infectious agent comprisesa virus, a bacterium, a parasite, or a prion.
 12. The method of claim 11wherein the virus is an influenza virus.
 13. The method of claim 11wherein the virus is a coronavirus.
 14. The method of claim 13 whereinthe virus is a SARS coronavirus.